1. Field of the Invention
The present invention relates to a process for rapid isolation and purification of yolk antibodies, in particular IgY antibody, from anseriform bird yolk, and the yolk antibodies obtained thereby. More particularly, the present invention relates to a process for isolation and purification of yolk antibodies from anseriform bird yolk by an adsorption chromatographic procedure using a porous water insoluble non-charged adsorbent to accomplish a desired separation of yolk antibodies, and by a salting-out procedure that differentially precipitates the IgY antibodies. The present invention also relates to uses of the IgY antibodies in quantitative or qualitative immunoassay or in the preparation of pharmaceutical compositions directing to an etiological agent of interest.
2. Description of the Related Art
Antibodies are used widely in many biological investigations and clinical applications. Sera obtained from hyperimmunized mammalians are the most common source of polyclonal antibodies. Antibodies derived from such immune sera belong to a group of proteins called “immunoglobulins,” among which the immunoglobulin G (IgG) is the most abundant. The IgG molecule consists of three domains, namely two Fab regions and one Fc region. The Fab portion involves mainly in antigen binding. The Fc portion, though having no ability to bind with an antigen, directs several biological activity of an antibody, such as complement fixing and Fc receptor binding.
In the art of immunodiagnostics, an intact IgG molecule is not suitable for use in detection systems and immunological assays involving mammalian sera since the Fc region on an IgG molecule is capable of binding to Fc receptors, activating the complement system, and reacting with rheumatoid factor in mammalian sera. Removal of the Fc portion of an IgG molecule frequently leads to a reduction in the interference (E. Lamoyi, Methods in Enzymology 121:652-663, 1986).
Some of the suggested uses of antibody in immunotherapy include treating patients with intoxicated bacterial toxins or snake venoms (see, for example, U.S. Pat. No. 5,340,923 and U.S. Pat. No. 5,601,823), and protection of neonatal piglets against fatal enteric colibacillosis (see, for example, H. Brussow et al., J. Clin. Microbiol. 25:982, 1987; and C. O. Tacket et al., New Eng. J. Med. 318:1240, 1988). Since the Fc fragment of an antibody molecule is known to be the most antigenic portion of the immunoglobulin (E. M. Akita et al., J. Immunol Methods. 162:155-164, 1993), cleavage of the same which results in the formation of an F(ab′)2 fragment will reduce significantly a number of potential allergenic sites on the immunoglobulin molecule and is thus beneficial to human or animals administered with the immunoglobulin.
Recently, the divalent F(ab′)2 antibody fragment has been shown to be more useful in the immunodiagnostic tests (M. Muratsugu et al., J. Colloid Interface Sci 147:378, 1991; and J. L. Ortega-Vinuesa et al., J. Immunol Methods 90:29, 1996) and more suitable for development of the immunoassays involving mammalian sera than the parent IgG.
The F(ab′)2 antibody fragment, however, has not found widespread use in clinical immunodiagnostic kits as one might expect. This may be attributed to the difficulties and cost-ineffectiveness of large scale production of the F(ab′)2 fragments, which is conventionally made by pepsin digestion of IgG and subsequent purification via chromatography.
Ducks and their phylogenetically close relatives and some reptiles, such as turtles, have three kinds of serum immunoglobulins: a macromolecular immunoglobulin IgM (800 kDa in duck), and two isoforms of low molecular weight IgG with sedimentation coefficients of 7.8 S (in duck, 180 kDa) and 5.7 S (in duck, 130 kDa), respectively. (E. R. Unanue et al., J. Exp. Med. 121:697-714, 1965; H. M. Grey, J. Immunol 98:811-819, 1967; and B. Zimmerman et al., Biochemistry 10:482-448, 1971). Avian IgG is oftentimes called IgY due to their existence in egg yolk besides in sera. The 5.7 S IgY, constituted with shorter heavy chains, is structurally and antigenically similar to the F(ab′)2 fragment of the 7.8 S IgY (FIG. 1), and this fact leads to the nomenclature of IgY (equivalent to 7.8 S IgY) and IgY(Δ Fc) (equivalent to 5.7 S IgY) to represent both isoforms of IgY (K. E. Magor et al., J. Immunol. 149:2627-2633, 1992).
Studies conducted in the infected or experimentally immunized birds showed that duck antibodies are deficient in a number of biological effector functions, including complement fixation and Fc receptors binding, without sacrificing their binding activity to corresponding antigens (G. W. Litman et al., Immunochemistry 10:323, 1973; and T. E. Toth et al., Avian Dis. 25:17-28, 1981). This may reasonably result from the apparent lack of Fc-equivalent region of the IgY(Δ Fc) antibody that constitutes the quantitatively major component of anseriform bird antibody response. It is thus believed that the IgY(Δ Fc) antibody, which appears to be a structural and functional analog of the F(ab′)2 fragment, would provide magnificent advantages in immunological uses, if a promising process for manufacturing the antibody could be found, and the appropriate physical requirements for its activity could be identified.
Avian yolk antibodies have been reported to exhibit useful properties for both research and clinical applications as mammalian antibodies do (see, for example, U.S. Pat. No. 5,340,923; U.S. Pat. No. 5,585,098; U.S. Pat. No. 5,601,823; and U.S. Pat. No. 5,976,519). Egg yolks derived from a laying hen is inexpensive and more convenient and safer to handle as compared to the hyperimmunized mammalian sera. More importantly, yolk antibodies are able to stand up to the scrutiny under modern animal protection regulations (A. Poison et al., Immunol. Commun. 9:475, 1980; and B. Gottstein et al.). These facts suggest a potential use of egg yolk as a commercial source of antibodies.
However, high contents of lipidic substances, such as fatty acids, cholesterol and lecithin, in egg yolk make the isolation of yolk antibodies a cumbersome and laborious task. Many efforts have been made in this regard. For instance, water soluble precipitants, including agar, pectin (Japanese Kokai No. 64-38098 published in Feb. 8, 1989), dextran sulfate (J. C. Jensenius et al., J. Immunol. Methods 46:63, 1981), natural gums (H. Hatta et al., J. Food Science 53:425, 1988) and polyethylene glycol (PEG) (A. Poison et al., Immunol. Invest. 14:323, 1985; see also U.S. Pat. No. 4,550,019 issued to A. Poison) were used to precipitate non-aqueous bio-molecules, mainly lipids and yolk granules, to thereby harvest a water soluble phase containing abundant yolk antibodies of the entire population. A. Hassl et al. developed a two-step chromatographic process, comprised of hydrophobic interaction chromatography and size exclusive chromatography, for further isolation of yolk antibodies of the entire population from a PEG-purified fraction (A. Hassl and H Aspock, J. Immunol. Methods 110:225, 1988). Akita et al described an improved method for isolating IgY, in which yolk antibodies were extracted from chicken eggs by diluting the egg yolks with a large volume of water and subjecting the resultant supernatant to size exclusive chromatography and/or ion exchange chromatography (E. M. Akita et al., J. Immunol. Methods. 160:207, 1993; and E. M. Akita and S. Nakai, J. Food Sci. 57:629, 1993).
However, all these studies and patents focus on the isolation of the entire population of yolk antibodies (which at least includes IgY present or absent Fc region) from avian eggs, rather than on the purification of IgY(Δ Fc) and IgY antibodies selectively. Moreover, since IgY(Δ Fc) antibodies are present only in birds belonging to the Order Anseriformes, including duck and goose, and since the lipid content in the egg yolk of the anseriform birds is reported higher than that in the galliform birds, such as chicken and turkey, the conventional methods described above provide no suggestion of a successful purification of IgY(Δ Fc) antibody. IgY(Δ Fc) antibody was only purified by co-precipitating with IgY from duck serum (D. A. Higgins et al., Veterinary Immunology and Immunopathology 41:169-180, 1995) with complexes and expensive procedures, but still no IgY(Δ Fc) antibody alone was selected isolated from egg yolk.
Therefore, there exists a need for a rapid, cost-effective and high-throughput process that provides easy isolation of the desired IgY antibody from the antibody pool of anseriform bird egg while maintaining the activity of the IgY antibody. The substantially purified IgY(Δ Fc) antibody may acts as a new type of F(ab′)2 antibody for various immunodiagnostic and immunotherapeutic uses.